Accessing the nucleus through the nuclear membrane poses one of the major obstacles for any therapeutic molecule that is large enough to be excluded due to nuclear pore size limits. Non-viral vector development for gene therapy is a field where mechanisms for improving nucleic acid delivery to the nucleus have been studied but only limited success has so far been achieved. Besides the large size of some nucleic acids, like plasmid DNA, hampering their access to the nuclear compartment, also for small oligonucleotides, the less time they reside in the cytoplasm the less prone they are to degradation processes. Moreover, it also should be beneficial if higher nuclear concentrations could be achieved in some applications. The use of peptide nuclear localization signals (NLS) together with nucleic acids has been one of the most explored formats in trying to achieve better nuclear transfer of genetic material.
Nucleocytoplasmic transport of endogenous molecules is a regulated process where small molecules are able to diffuse through the nuclear pore complex of the nuclear membrane while molecules >40 kDa require the use of a signal and energy-mediated processes. Import of nuclear proteins requires nuclear localization signals (NLS) in the form of specific amino-acid sequences which mediate the interaction with carrier proteins [1]. The best studied NLS sequence is the SV40 large-T antigen. It mediates the interaction between the cargo protein, bearing the NLS signal, and an import receptor consisting of an adaptor protein, importin alpha, which directly binds the NLS signal, and importin beta which is the mediator of the actual import process through the nuclear pores [2].
An archetypical NLS, which is mediating the interaction with several different types of import receptors, is found on the ribosomal L23a protein. This NLS, which has a higher degree of complexity and harbours very basic regions, is thought to have evolved prior to the evolutionary divergence of import receptors [3].
In addition to the cargo and import receptor interactions there are other factors needed in the process of nuclear import. It is the asymmetric distribution of the factors Ran, RCC1, RanGAP1, RanBP1, creating a steep RanGTP gradient across the nuclear envelope that allows the directional movement of the cargo-importin receptor complex to the nucleus [4, 5].
Besides nuclear proteins some RNAs, in the form of small nuclear ribonuclear protein complexes (snRNP), use signals for nuclear import. These RNAs are comprised of the major spliceosomal U snRNAs, such as U1, U2, U4 and U5 and are the major building units of the spliceosomal complex. U snRNAs are transcribed in the nucleus by RNA polymerase II after which they acquire a 7-methylguanosine (m7G) cap structure at their 5′ end. This cap structure acts as a nuclear export signal that is recognized by the cap-binding complex (CBC). The CBC complex is in turn recognized by the export receptor CRM1 with the help of PHAX adapter leading ultimately to the nuclear export of the U snRNA [6].
After release in the cytoplasm the U snRNA is recognized by the survival of motor neuron complex (SMN) that directs the proper assembly with a group of Sm proteins [7-10]. Subsequently the m7G cap is hypermethylated to a trimethylguanosine (m3G) cap structure (FIG. 1) by the small-nuclear-RNA cap hypermethylase [11, 12]. The matured snRNP is then imported back into the nucleus.
This nuclear transport involves two different pathways and two very distinct import signals, both of which, however, recruiting importin beta [13-16]. The first pathway uses a, still poorly defined, import signal present in the Sm core domain of the snRNP formed by the Sm proteins [17]. The second pathway involves the use of the 5′ 2,2,7-trimethylated guanosine (m3G) cap structure [18]. The m3G-CAP signal is recognized by the import adaptor protein snurportin (SPN1) [14, 19, 20], which in turn is recognized by importin beta [14-16, 21].
Nuclear import of therapeutic molecules is of great importance for many applications. For example, specific targeting of exogenous proteins, such as antibodies, to the nucleus for the purpose of radioimmunotherapy, is seen as a way to increase cytotoxicity effects in cancer cells [22]. In particular, one of the areas where nuclear targeting has been getting a lot of attention is the gene delivery/gene therapy field. This is especially true in non-viral or synthetic vector development, since these transport systems need to mimic most of the virus strategies used to overcome several cellular barriers of which the nuclear membrane is the ultimate one.
Some approaches to nuclear delivery of nucleic acids, like plasmid DNA, have relied on the DNA sequence itself [23]. A plethora of other types of strategies for nucleic acid delivery has been based, however, on the direct or indirect attachment of NLS peptides to the nucleic acid molecules as a way to promote binding of the importin alpha/importin beta heterodimer to the nucleic acid construct and its subsequent nuclear translocation [24]. The SV40 large T antigene NLS (especially in its shortest form pkkkrkv) has been one of the most employed NLS peptide. It has been associated to DNA via ionic interactions [25]; via chemical coupling [26]; through the use of peptide nucleic acids (PNA) linked to the NLS and bound to DNA in a sequence specific manner [27, 28]; through the use of a triple helix forming oligonucleotide system [29] and by the use of biotinylated DNA bound NLS conjugated streptavidin [30]. An example of another peptide NLS used is the non-classical NLS defined by the M9 sequence, coming from the heterogeneous nuclear ribonucleoprotein (hnRNP) A1, which has been used together with a DNA binding peptide for increased nuclear delivery after lipofection of non-dividing cells [31].
Current means to transport macromolecules, e.g. nucleotides, for example using classical positively charged peptide-based NLS sequences, are not very efficient and there is a need for novel complexes and methods for transmembrane transport with improved specificity, transport capacity and stability.
WO 00/04144 discloses the m3G-CAP specific nucleus import receptor protein and the production and use thereof. The disclosure of WO 00/04144 focuses on the polypeptide, antibodies against it, and nucleic acids encoding the same.